1. Field of the Invention
This invention relates to the field of mammalian ouabain-like factors (OLF) and more particularly to a novel dihydroouabain like factor (Dh-OLF), and its use for the detection and treatment of conditions and diseases associated with regulation of the sodium-potassium pump, in particular for the detection and treatment of congestive heart failure. This invention also relates to those plant cardenolides that are cardiac glycosides. This invention also relates to antibodies and antibody fragments useful for the treatment of toxicity due to excess OLF or Dh-OLF.
2. Description of the Background
Digoxin and ouabain, plant cardenolides that are the two most commonly used cardiac gylcosides, are commonly administered to patients suffering from congestive heart failure because of their beneficial effect on cardiac contractility, that is, their positive inotropic effect. Positive inotropic effect generally refers to the enhancement of contractility of the cardiac cells in a dose-dependent manner. These drugs produce an increase in the force and velocity of ventricular contraction, and also a slowing of the heart rate. These two effects combine to provide a stronger heart beat. However, cardiac glycosides have narrow therapeutic indices and their use is frequently accompanied by toxic effects that can be severe or lethal.
The exact mode of action of cardiac glycosides is not completely known, but it is thought that the effects are mediated through regulation of the sodium pump. Abnormal sodium pump activity has been postulated to be involved in the pathophysiology of several diseases, including cardiovascular, neurological, renal, and metabolic disorders, among others. These complex effects may be related to the role of the pump in controlling the cellular ingress of other molecules.
The NA+,K+-ATPase enzyme or sodium pump is a membrane protein responsible for establishing an electrochemical gradient of Na+ and K+ ions across the plasma membrane of mammalian cells. The ion gradient formed by this enzyme is necessary for the active transport of essential nutrients into the cells, for regulation of osmotic balance and cell volume, and for maintaining the resting membrane potential in excitable cells. The Na+,K+-ATPase enzyme is the only known receptor for cardiac glycosides such as digitalis. The tight conservation of the digitalis binding site over many phyla, among other observations, suggests the existence of endogenous sodium pump inhibitors (SPIs) in mammals as well. These hypothetical mammalian inhibitors would be involved in modulating the activity of the sodium pump, and might be involved in vivo sodium homeostasis.
One of the side effects of administration of cardiac glycosides is arterial hypertension. It is thought that an excess of endogenous factors may also cause arterial hypertension, which is a risk factor in complications associated with various organs. Higher plasma levels of ouabain-like compounds (OLCs) were found in patients with primary aldosteronism and ectopic corticotrophin syndrome, two types of mineralocorticoid hypertension. In addition, 30-45% of patients with essential hypertension had increased plasma levels of OLCs and the blood pressures were statistically correlated with the OLC levels. Although presently available anti-hypertensive agents have proven beneficial, they still fall short of completely reversing the effects caused by high blood pressure. Accordingly, there still is a need for agents which are more specific and effective than those presently available in order to reverse the toxic effects of high levels of ouabain, whether administered or endogenous. It would be of further benefit to have a means of predicting the occurrence of hypertension in patients before the occurrence of organ damage due to sustained hypertension.
The kidneys have also been implicated in changes in blood pressure variations. A rise in blood pressure has been observed in clinical forms of hypertension, such as Liddle""s syndrome, glucocorticoid-suppressible aldosteronism, and the syndrome of apparent mineralocorticoid excess, which appears to be closely related to a constitutive increase in sodium (Na+) reabsorption in the kidney. Essential hypertension is a heterogeneous disease which is thought to result from genetic and environmental factors that interact to increase blood pressure, thus, gene mutations may also contribute to hypertension, and since these genes were shown to converge on a final common pathway, the result may be increased Na+ reabsorption and/or decreased Na+ excretion in the kidney. Although still uncertain, however, why increased Na+ reabsorption might lead to high blood pressure, salt retention and plasma volume expansion trigger the secretion of sodium pump inhibitors (SPIs), which restore extracellular fluid volume via natriuresis. An increased secretion of SPIs, thus, may also elevate cytosolic Ca2+ and produce vasoconstriction, which will account for the development and perpetuation of hypertension. Whatever the mechanism, it has been empirically noted that hypertension is associated with abnormal function of sodium regulation.
The chronic administration of ouabain has been shown to cause the development of hypertension in rats. Immunization of these Dahl salt-sensitive (S) rats against ouabain prevented a reduction in renal mass-saline hypertension and Na+-induced hypertension.
Plant cardenolides, such as ouabain and digoxin, have been shown to bind specifically to highly conserved epitopes on the xcex1-subunit of the Na+,K+-ATPase enzyme (sodium pump) and to stabilize the phosphorylated intermediate. This effect leads to the inhibition of pump-associated transport of sodium, potassium and other important biologic compounds across cell membranes. It has furthermore been postulated that a hormonal-axis may regulate the activity by mammalian ligands similar to plant cardenolides of the sodium pump. Two types of mammalian compounds have been found to date with properties similar to those of plant cardenolides: (1) digoxin-like factors such as DLF or DLIF, and (2) ouabain-like factors such as OLF or HIF. The DLIF family includes a series of deglycosylated species and Dh-DLIF, a dihydrodigoxin-like isomer. Cytochrome P450 was recently shown to mediate the conversion of dihydrodigoxin, a plant cardenolide of low DLIF immunoreactivity, to one with high DLIF immunoreactivity. This suggests a possible in vivo metabolic conversion of Dh-DLIF, a less active dihydro-species, to DLIF, a more biologically active species. The present inventors have recently reported the presence of a deglycosylated species of human OLF analogous to the plant ouabain. Dihydroouabain (dho), the chemically-reduced form of ouabain having a saturated lactone ring, has been used to study the biological activity of ouabain and its interaction with the sodium pump. The chemical formulas of ouabain and dihydroouabain are shown below.
Ouabain Dihydroouabain
C29H44O22 C29H46O12 
MW 584 MW 586
Ouabain contains an aglycone consisting of a steroid nucleus with an unsaturated lactone ring attached at the C17 position, a sole sugar molecule, a rhamnose, attached at the C3 position. The only difference between ouabain and dihydroouabain (dho) is that the latter has a fully hydrogenated lactone ring.
In general, the reduced lactone ring forms of both digoxin and ouabain show relatively lower potencies than their oxidized counterparts for inhibiting Na+,K+ pump catalytic activity. However, they have other biophysical properties, such as being rapidly washed out from tissues, etc. that are rather advantageous.
Antibodies to specific molecular structures may be made by immunization of animals such as mice or rabbits with the target structure by methods known in the art. Non-human antibodies, including polyclonal and monoclonal antibodies, raised against specific human molecules have been used for diagnosis and therapy as is known in the art. Chimeric antibodies are direct fusions between variable domains (the reactive portion or complementary determining region or CDR) of one species and constant domains (framework region, FR) of another. Murine/human chimeric antibodies, in which mouse CDR is fused to human FR, have been shown to be less immunogenic in humans than whole murine antibodies. The size of the bioactive molecule may also be reduced, so as to increase the tissue target availability and to allow for ultimate excretion through the kidneys of the antibody, by removing most of the heavy and light chain constant regions to form an Fv antibody, which is basically an isolated CDR. Although any species may be used for the production of antibodies, when large quantities of antibody are required, it is advantageous to use large animals such as sheep, goats or horse. However, any mammal may be used. Antibodies or fragments from either polyclonal or monoclonal sources may be purified by affinity separation. Common to all of these potentially therapeutic forms of antibody are the required CDRs, which guide the molecule to its ligand.
The development of immunoassays has permitted the utilization of antibodies for diagnostic purposes, in particular, for the determination of levels of different endogenous agents in animal and human tissues. Immunoassays rely on the quantitative binding of a known amount of a known antigen to a known amount of antibody, on the binding of this antibody to the antigen to be used as a standard, and on a comparison of the latter to the amount of unknown analyte in a sample which is bound by the same antibody. When both reactions are conducted in the same medium, analyte present in the unknown sample interferes with the binding of known quantities of antibody to known quantities of antigen. A key step of these assays is the separation of bound antibody or antigen from the unbound antibody or antigen. Many configurations for this reaction are well known, either as direct immunometric, competitive or displacement assays, and the like. Quantitative results are generally obtained through hemagglutination assays, radioimmunoassays, enzyme-linked assays, and the like.
In an immunoassay, a given analyte present in a animal or human tissue is compared to a known quantity of the solubilized standard analyte. The most common tissue analyzed is blood, and more specifically serum and/or plasma from blood, but urine, cerebrospinal fluid, different serum preparations and different animal and human tissues and fluids are also routinely assayed. In endocrinology and clinical chemistry, enzyme-linked assays and radioimmunoassays have been used to determine levels of hormones, proteins and lipid metabolites, among other substances. Levels of certain molecules in the blood or other tissue may be indicative of a disease state, often at a very early stage before symptoms are manifest. These molecules are termed markers of the condition.
Accordingly, there still exists a need for an assay to detect the presence of markers indicative of sodium pump function, particularly such an assay that is fast, cost effective and which may be conducted without highly specialized personnel. This technology would be immediately applicable to the screening of populations at risk as well as of patients undergoing treatment for diseases requiring control of sodium pump function.
This invention relates to a purified, isolated mammalian dihydroouabain-like factor (Dh-OLF) that inhibits the sodium pump (Na+,K+-ATPase), and is a pro-drug for the mammalian ouabain-like factor (OLF), to which it is converted in vivo. Conversely, OLF is a pro-drug for Dh-OLF, to which it is converted in vivo. Although Dh-OLF has an hydrogenated lactone ring, as does the plant derived dihydroouabain (dho), the factor of the invention has other characteristics which set it apart from dihydroouabain. The Dh-OLF has a molecular weight of about 586 and substantially fails to cross-react with mammalian ouabain-like Na+,K+-ATPase inhibitory factor (OLF) for binding to anti-ouabain antibody. It is significant that it cross-reacts with plant dihydroouabain (dho) for binding to anti-dho antibody. The deglycosylated compounds show a similar immunoactivity pattern as the sugar-containing factors, suggesting that the major antigenicity resides in the lactone moiety. Therefore, for purposes of describing this invention, OLF, Dh-OLF, ouabain and dho are intended to include their deglycosylated analogues. xe2x80x9cDhoxe2x80x9d is comprised of isomers dho-A and dho-B. Dh-OLF has maximal uv absorbance at 196 nm, a non-peptidic chemical structure as evidenced by lack of absorbance at 280 nm, a non-lipidic chemical structure as evidenced by lack of lipase digestion and lack of staining with a lipid stain, a fully hydrogenated lactone ring; 10-fold lower potency than OLF and 3-fold higher potency than plant dihydroouabain-B (dho-B) for inhibiting Na+,K+-ATPase catalytic activity, 10-fold lower potency than OLF and 3-fold higher potency than plant dho-B for phosphorylating Na+,K+-ATPase xcex1 subunits, a concentration-dependent inhibition of Na+,K+-ATPase catalytic activity; a concentration-dependant phosphorylation of Na+,K+-ATPase xcex1 subunits, and a high pressure liquid chromatography elution pattern about the same as dihydroouabain.
The endogenous ouabain-like factors (OLF and Dh-OLF) may be isolated by a method involving the use of uv spectrophotometry, preparative high pressure liquid chromatography or affinity separation with antibodies or other binding agents that are specific to them or to ouabain or dihydroouabain.
This invention also relates to monoclonal and polyclonal antibodies and fragments thereof and to their use for the detection of Dh-OLF and/or OLF in biological (animal) samples to make an early assessment of disease states associated with abnormal levels of Dh-OLF or abnormal ratios of Dh-OLF to OLF. The antibodies or, preferably, fragments thereof, are also suitable for therapeutic use in the treatment of various diseases associated with increased levels of Dh-OLF, including metabolic diseases, heart disease, high blood pressure, renal impairment, neurologic disorders such as Alzheimer""s disease, psychiatric conditions, ophthalmic diseases, and sexual dysfunction, among others. Monoclonal antibodies, if desired, may also be made by methods well known in the art.
In addition, this invention also provides an in vivo method for reducing levels of of Dh-OLF or OLF by administration to a subject in need of the treatment of a prophylactically or therapeutically effective amount of the antibody of the invention or an anti-dho antibody or, preferably, fragments of these antibodies. Other specific binding agents are known, such as synthetic compounds such as aptomers. All such specific binding agents which bind ouabain or dihydroouabain or their endogenous analogues can likewise be use in this method.
The levels of Dh-OLF and/or OLF present in a test sample may be assessed with the aid of a diagnostic assay of this invention that relies on the selective binding of the factor by an antibody having affinity for this compound or dho isomer mixture, and the subsequent addition of a labeled antibody Fc-binding agent to generate a signal which is linearly correlatable with the amount of Dh-OLF present in the biological sample.
The levels of Dh-OLF and/or OLF present in a test sample may alternatively be assessed by quantitative HPLC, where the elution peaks are compared to the peaks derived from a known amount of Dh-OLF and/or OLF. The hydrogenation of ouabain using palladium catalyst at 50xc2x0 C. produces two isomers (dho-A and dho-B). The elution peaks thought to be Dh-OLF of a test sample may be compared to the peaks derived from a known amount of dho-B and the concentration of Dh-OLF estimated from comparison of the respective peak heights.
It is known that the ratio of OLF to Dh-OLF or OLF to Dh-OLF may vary from subject to subject. This variation of ratio is thought to be due to a variable rate of interconversion of Dh-OLF and OLF. Since OLF has ten-fold the activity of Dh-OLF on the activity of the sodium pump, the conversion enzyme(s) will provide a novel target for the development and testing of antihypertensive or antihypotensive drugs.
A more complete appreciation of the invention and many of the attendant advantages thereof will be readily perceived as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying figures. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that certain substitutes and modifications may be made without departing from the spirit and scope of this invention as defined by the appended claims.